Devices and methods for the synthesis of nucleic acids

ABSTRACT

Cylindrical devices (frits) are prepared by embedding aminoalkyl- or mercaptoalkyl-modified Controlled Pore Glass (CPG) in high-density polyethylene. Methods and devices pertaining to their use in the synthesis of nucleic acids are described. A reusable synthesis column or a reusable 96-chamber synthesis plate have been designed to hold one to 96 of the said frits that are inserted reproducibly into the synthesis chambers with a frit insertor. A short gas surpressure is required to drive entry of chemical reagents into the said frit. Reagents are retained into the frit until a second, longer surpressure is applied to drain the said reagents.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional application of pending U.S. patent application Ser.No. 10/776,694 filed Feb. 12, 2004.

BACKGROUND OF THE INVENTION

Composite structures made of thermoplastic resins and particulatematerials have been widely described. U.S. Pat. No. 4,153,661 describesa method for making composite sheets comprising particulate distributedin a matrix of polytetrafluoroethylene (PTFE) fibrils. U.S. Pat. Nos.4,373,519 and 4,565,663 disclose methods for making water-swellablecomposite sheets having hydrophilic absorptive particles enmeshed in aPTFE matrix. U.S. Pat. No. 4,971,736 describes methods of enmeshingnon-swellable particulate in a PTFE matrix and their use aschromatographic articles.

U.S. Pat. No. 5,904,848 describes methods for calendering and sinteringan aqueous dispersion of PTFE and controlled pore glass (CPG) into rigidporous sheets of 5 to 200 mils in thickness from which disc membranesare being cut. The membrane porosity is adjusted by using CPG of variouspore sizes. Post-silanization treatment of the said disc membranes isrequired to introduce reactive moieties onto the CPG surface.

U.S. Pat. No. 6,416,716 disclosed methods to prepare tubes whichinterior surfaces are embedding separation medium particles. Forinstance a polypropylene tube filled with C-18 particles was heated toembed the separation medium particles into the interior of the tubes dueto the melting of the polypropylene. Other embedded devices described inWorld patent No. 00/21658 are prepared by sintering functionalizedpolystyrenes with polyalkylenes especially polyethylene andpolypropylene. The said devices contain at least 10 μmol of reactivefunctionalities available for synthetic purposes notably peptidesyntheses. The porosity of those devices to methanol at ambienttemperature and pressure is described as being at least of 0.2 mL/min.

Although the general concepts of embedded devices have been discussed,cylindrical devices prepared by embedding modified-CPG in polyalkyleneand the methods pertaining to their utilization in the synthesis ofnucleic acids have not been developed thus far.

BRIEF SUMMARY OF THE INVENTION

The present invention described the preparation of cylindrical devicescalled frits, made from polyalkylene embedded modified-CPG. The saidfrits are prepared by embedding modified-CPG such as aminoalkyl-CPG ormercaptoalkyl-CPG into a polyalkylene network, providing a generallyuniform dispersion of the inorganic material into the resin. To be usedwith current nucleic acid synthesizers, the said frit must contain lessthan 10 μmol of reactive amino or mercapto moieties, preferably lessthan 2 μmol and especially less than 1 μmol. Entry and draining ofchemical reagents into and from the frits of the invention are broughtabout by applying a differential pressure such as a vacuum or preferablya gas surpressure on an automated synthesizer. Thus at ambient pressure,the porosity of those devices is such that the gravity-induced entry ofthe said chemical reagents is prevented. This allows for an efficientpre-mixing of reagents prior to their entry into the frit. Reagents arepushed into the frits by applying a short gas surpressure and areretained into the frits for the desired amount of time without dripping.This particular feature minimizes the volume of reagents required forthe synthesis to the void volume of the cylindrical frit, thereforeoptimizing the consummation of the said reagents and their reactivityprofile.

It is a further object of this invention to provide accessoriesfacilitating the use and the post synthesis manipulations of the saidsynthesis frits. To this purpose, synthesis plates have been drilledwith open top and bottom synthesis chambers to hold up to 96 fritz. Forlow throughput synthesis, single synthesis column with open top andbottom ends have been prepared. A frit insertor is used to insert fritsinto the synthesis columns or the plate chambers from their top ends.Upon completion of a synthesis, a frit extractor is used to push thefrits through the bottom ends of the said chambers or said columnswithout damaging frits, columns or chambers. This allows synthesisplates and synthesis columns to be reused.

It is still a further object of this invention to provide a reliablemethod enabling the automated synthesis of nucleic acids by using fritsthat have been derivatized with a catechol-based universal linker. Bydefinition, a universal linker allows the synthesis of nucleic acids ona solid support regardless of the nature of their 3′-terminal base byreacting with the 3′-end of a nucleoside, functionalized in particularwith a phosphoramidite moiety. The oligonucleotide-bound solid support,upon treatment under the usual conditions of deprotection, is recoveredas a 3′-hydroxyoligonucleotide.

The term oligonucleotides and nucleic acids refer to ribonucleic acidsor deoxyribonucleic acids in which modifications can take place at thelevel of the base, the ribose rings or the internucleotide phosphatebonds in a chemically known manner.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a top and bottom view of a 96-chamber synthesis plate.

FIG. 2 is a bottom view of a 96-chamber synthesis plate.

FIG. 3 is an enlarged, cross sectional view of a single chamber.

FIG. 4 is a cross sectional view of a synthesis column.

FIG. 5 is a schematic view of an 8-pin insertor.

FIG. 6 is a schematic view of an 8-pin extractor.

FIG. 7 is a schematic view of a combo 1-pin insertor/1-pin extractor.

FIG. 8 is a schematic view of loading and extracting frits from a96-chamber synthesis plate with an 8-pin insertor and an 8-pinextractor.

FIG. 9 describes the preparation of an embedded catechol-based universalsupport.

DETAILED DESCRIPTION OF THE INVENTION A. Frit Preparation

To produce the frits of the invention, silane-modified CPG isadvantageously used in order to control the said frit loading capacityprior to its manufacture. Bifunctional silanes, having a firstfunctional group enabling covalent binding to the glass surface (aSi-halogen or Si-alkoxy group) and a second functional group thatimparts the desired chemical modifications to the surface, are used tomodify the CPG surface. Silane-modified CPG are controlled porous glassbeads, which have been preferentially modified withaminoalkyl-trialkoxysilane, [alkylamino]alkyl(trialkoxy)silane ormercaptoalkyl(trialkoxy)silane and mixtures thereof. Preferentially,alkyl is selected from the group consisting of methyl, ethyl and propyland wherein alkoxy is selected from the group consisting of methoxy,ethoxy and propoxy. In a preferred embodiment, low loading capacity ((5to 30 μmol/g)aminopropyl-CPG 1 is prepared by reacting CPG (500, 1000 or2000 A pore diameter, preferably 1000 A, particle size 40/75 or 75/200microns, preferably 75/200) with aminopropyltriethoxysilane indichloromethane at room temperature.

A silane-modified CPG or a blend of two different silane-modified CPG ismixed with an aqueous-free polyalkylene in a solid weight ratio of 30 to50%. Polyalkylenes are selected from the group consisting of ultrahighmolecular weight polyethylene, high density polyethylene, medium densitypolyethylene, low density polyethylene, polypropylene, and mixturesthereof. Preferentially, aminoalkyl-CPG 1 is mixed in a solid weightratio of 35 to 45% with high-density polyethylene.

An aluminum plate drilled with 50 to 5000 wells, preferably 1000 to2000, is filled with the said polyethylene/silane-modified CPG mixture.In one embodiment, the aluminum plate dimensions (X, Y, Z in inch) arerespectively (14.0, 6.0, 0.50). Preferably, the said wells have a roundcross sectional shape. In one embodiment, cylindrical wells with adiameter/length (in mm) of 3.90/6.0 or 3/90/9.0 or 3.90/12.0 have beendrilled. Those wells yield cylindrical frits which sizes are optimal tocontain 50 nmol, 200 nmol and 1 μmol of reactive moieties, respectively.

The said filled aluminum plate is heated at approximately 180 to 200° C.under a normal atmosphere for a predetermined time (5 to 20 min).Heating schedule is a function of the mixture composition, the size ofthe aluminum plate and the number of chambers. At these temperatures,around 1 to 5% shrinkage uniformly occurs throughout the structure. Foruse with this invention, preferably the firing schedule, temperature andpowder composition can be modified in such a way as to significantlycontrol shrinkage. Upon cooling the aluminum plate, the frits areremoved from the wells and are controlled for adequate mass anddiameter.

B. Accessories

It is a further object of this invention to provide accessories enablingconvenient and reproducible uses of the synthesis frits.

Synthesis plates have been prepared and used as frit holders to carryout the high throughput synthesis of nucleic acids. The said plate ispreferably made of Teflon. Preferably, the plate surface is modeled offthe industry standard. This way, equipment such as multiple pipetters orrobots designed for use with 96-well plates may be easily adjusted foruse with the said synthesis plate. The synthesis plate may be of anyheight (Z), preferably between 1.5 and 2.0 inches. In one preferredembodiment, the plate dimensions (X, Y, Z) in inch are 4.98, 3.35, 1.60,respectively.

Any number of cylindrical open top and bottom ends chambers may bedrilled into a synthesis plate. Preferably, the number of chambers is amultiple of 48 (i.e., 96, 384, 1536), especially 96 (see FIG. 1).Preferably, the spacing between chambers, both in the X and Y directionof the plate, is modeled off the industry standard 96-well plate (8×12mutually perpendicular rows).

FIGS. 2 and 3 show a cross-sectional view of a 96-chamber plate and anenlarged cross-sectional view of a single chamber, respectively. Achamber is made of a top cylinder, a middle cylinder and a bottom cone.The sidewalls of the top and middle cylinders may be of any height,depending on the desired volume of reagents per chamber. Preferably, theheight of each cylinder is between 0.40 and 0.80 inch. The top andmiddle cylinder cross diameters are wider than the cross diameter of acylindrical frit. Preferably, the top cylinder is wider by 0.10 to 0.15inch and the middle cylinder is wider by 0.01 to 0.05 inch. The bottomcone or frit holder has a cross diameter smaller than the cross diameterof a cylindrical frit. Preferably, the cone has a top cross diameter0.001 to 0.003 inch smaller than the cross diameter of a cylindricalfrit, preferably 0.002 to 0.003 and a bottom cross diameter 0.003 to0.008 inch smaller than the cross diameter of a cylindrical frit,preferably 0.004 to 0.005 inch. A smaller diameter cone allows the fritto be held tightly which has a three-fold effect: (i) it prevents thefrits from being extracted from the synthesis plate during the automatedsynthesis of nucleic acids when a gas (nitrogen, argon) surpressure isapplied to drive the chemical reagents into the frits or to drain thereagents from the frits. (ii) It prevents dripping and draining of thereagents along the chamber sidewalls. Thus, it ensures that the reagentsare forced into the frits or are fully drained when a gas surpressure isapplied. (iii) It maintains a homogenous backpressure from eachfrit-filled chamber regardless of its synthesis status (i.e. synthesiscompleted or not in the said chamber).

For low throughput nucleic acid synthesis, single synthesis columnsprepared by injection molding of polypropylene are used. The saidcolumns are opened cone with open top and bottom ends (FIG. 4). They areused to hold a single frit in a low throughput synthesis of nucleicacids. Notably, the said column has a holding cylinder 0.002 to 0.010inch smaller than the cross diameter of a cylindrical frit, preferably0.002 to 0.004 inch.

A one- to 96-steel pin insertor is used to insert from one to 96 fritsinto the synthesis chambers from their top ends and secured themreproducibly into the bottom cone of the chambers. Preferably, an 8-pininsertor is used to insert simultaneously eight frits into eightsynthesis chambers. A detailed schematic view of an 8-pin insertor isshown FIG. 5. The steel pin insertor length is slightly longer than thecombined length of the top and middle cylinders of a synthesis chamber.

Upon completion of a synthesis, a one- to 96-steel pin extractor is usedto extract one to 96 frits through the bottom ends of the synthesischambers. Preferably, an 8-pin extractor is used to extractsimultaneously eight frits from eight chambers into eight collectionvials. A detailed schematic view of an 8-pin extractor is shown FIG. 6.The steel pin extractor length is slightly longer than the synthesischamber length.

A schematic view of a combo 1-pin insertor/1-pin extractor is shown FIG.7. A 1-pin insertor/1-pin extractor is used to insert or extract asingle frit in/from a synthesis column or a synthesis chamber.

Pushing the frits through the narrower bottom end of the synthesischambers or the synthesis columns does not damage the frits or thesynthesis chambers or the synthesis columns. Therefore, the synthesisplates and synthesis columns are advantageously reused, contrarily tocurrently available consumable DNA synthesis columns. Another advantageis that the frits once extracted into collection vials or a 96-wellcollection plate are easily manipulated for post synthesis treatments.

C. Methods Describing the Use of the Synthesis Frits

To illustrate the use of the frits in the synthesis of nucleic acids,frits functionalized with a catechol-based universal linker have beenprepared from aminopropylCPG frits 2. Catechol-based universal linkershave been described in U.S. Pat. No. 6,590,092. They are usedirrespective to the first nucleotide of the said nucleic acids to besynthesized onto the solid support and irrespective of the type ofmonomer reagent used during the synthesis.

In a preferred embodiment, aminopropylCPG-frits 2 are reacted withexcess carbonate 3 (FIG. 9). Excess carbonate is used in order to ensurea complete reaction of the amino moieties. Disappearance of the aminogroups is monitored by ninhydrin test. The resulting carbamate boundcatechol (regioisomeric mixture, one isomer shown) and the remaining CPGsilanol groups are capped simultaneously with excesstrimethylsilylimidazole yielding frits 4.

Frits 4 are employed to synthesize nucleic acids on automatedsynthesizers using synthesis columns or preferably using a 96-chambersynthesis plate. A schematic loading of a 96-chamber synthesis platewith an 8-pin insertor is described in FIG. 8. The synthetic cyclebegins with a catechol deprotection step carried out with 3%trichloroacetic acid in dichloromethane, i.e. the reagent commonly usedin the 5′-detritylation step. The first nucleotide is then attached tothe catechol bound support using conventional phosphoramidite chemistryunder the same conditions and with the same monomer reagent as thecondensation of the second nucleotide with the desired first nucleotidebonded to the support. The said first nucleotide corresponds to thefirst nucleotide in the sequence of the said nucleic acid. Chainelongation occurs by sequential reaction of 5′-protected nucleosidephosphoramidites with the 5′-hydroxyl-end of the oligonucleotide boundpolymer. Oxidation (I₂/pyridine/acetonitrile/H₂O), capping (Ac₂O) anddetritylation (3% trichloroacetic acid in dichloromethane) steps arecarried out as usual.

After the reagents are delivered into the synthesis chambers or thesynthesis columns, a brief application of pressure is required to drivethe reagents into the frits. Indeed, at ambient pressure, a wetting of afrit is sufficient to prevent entry of chemical reagents. This allows anefficient pre-mixing of the chemical reagents such as activator and3′-phosphoramidite (or the synthesis columns) prior to their entry intothe frit. The reagents stay inside the frit as long as needed and areflushed when a full draining surpressure is applied. To deliver thereagents into the frits or drain the reagents, an optimal pressure of2.5 to 4.0 PSI at the chamber pressure is recommended. Delivery of thereagents into the frits requires a short pulse of pressure (one secondfor acetonitrile and dichloromethane solutions or two seconds fortetrahydrofuran solutions) while draining requires applying asurpressure for a longer time, at least 8 s and preferably 15 s.

Upon completion of a nucleic acid synthesis, a frit extractor is used topush down the oligonucleotide-bound frits without damaging them intovials or into a collecting 96-well plate (see FIG. 8). Thepost-synthesis cleavage of the oligonucleotide-bound CPG anddeprotection steps are carried out simultaneously by heating the fritswith 33% ammonium hydroxide (6 h at 80° C.), 40% aq. methylamine or aq.ammonia-methylamine (1:1, v/v)(4 h at 80° C.) to yield3′-hydroxyoligonucleotides free of any residual terminal phosphategroup. After discarding the frits, the basic solutions containing theoligonucleotides are evaporated.

The following examples illustrate the invention without limiting it:

Example 1 Preparation of 200 nmol Cathechol-Based Frit 4

A mixture of high-density polyethylene (66 g) and aminopropylCPG 1 (44g, 10 μmol/g, 1000-angstrom pore size, and particle size 75/200 microns)is prepared. The mixture is poured onto an aluminum plate drilled with1100 cylindrical wells. The well dimensions are diameter/length 3.90mm/9.0 mm, respectively. The plate is heated at 190° C. for 15 min andcooled before releasing the frits 2. Excess carbonate 3 is added to athousand frits suspended in dichloromethane under inert atmosphere atroom temperature. After gently stirring for 48 hours, the frits arefiltrated and washed successively with acetone and dichloromethane. Thefrits are resuspended in dichloromethane and trimethylsilylimidazole(0.80 mL) is added. After stirring for 2 hours, frits 4 are filtrated,washed with methanol and dichloromethane, and dried under vacuum.

Example 2 High Throughput Synthesis of 72 Oligonucleotides

Seventy-two frits 4 (200 nmol loading capacity) are inserted into 72chambers of a 96-chamber synthesis plate of the invention using an 8-pininsertor. All 24 unused chambers of the synthesis plate are sealed withduct tape. Oligonucleotides having three different lengths (25-mers,50-mers, and 75-mers) are synthesized on a high throughput synthesizer(BLP-192 from Biolytic Lab Performance, Ca) using conventionalphosphoramidite chemistry that is in current use and will thus be knownto those skilled in the art.

The following protocol is developed for a synthesizer using positivepressure for reagent delivery and draining. The gas pressure to drivethe reagents into the frits and to drain the reagents from the frits ismanually set at 2.5 PSI.

Line Description Time (sec) Volume (μl) Explanation 1 TCA delivery 150μl Deblock step 2 Drain 5 sec Pressure for draining 3 TCA delivery 150μl Deblock step 4 Push 1 sec Pressure to get the reagents into the frit5 Hold 5 sec Reaction time 6 Drain 15 sec  Pressure for draining 7 ACNdelivery 350 μl ACN delivery 8 Drain 25 sec  Pressure for draining 9Coupling Amidite and ETT deliveries 10 Push 1 sec Pressure to get thereagent into the frit 11 Hold 40 sec  Reaction time 12 Drain 5 secPressure for draining 13 Capping CAP A & B deliveries 14 Push 2 secPressure to get the reagents into the frit 15 Hold 10 sec  Reaction time16 Drain 8 sec Pressure for draining 17 Oxidation Iodine Delivery 18Push 2 sec Pressure to get the reagents into the frit 19 Hold 10 sec Reaction time 20 Drain 15 sec  Pressure for draining 21 ACN delivey 350μl ACN delivery 22 Drain 25 sec  Pressure for draining 23 Loop

The 200 nmol frit has a dead volume around 60 μl. To get the bestreaction yields with this frit, the total volume of reagents deliveredfor each step of the synthesis must be around 70-80 μl. To ensure acomplete DMT removal, the delivery of 2×150 μl of 3% TCA indichloromethane is recommended. Instead of using Tetrazol as activator,dicyanoimidazole (DCI) or ethyl thiotetrazol (ETT) is recommended for anoptimal coupling efficiency. Upon completing the syntheses, theresulting oligonucleotide bound frits are pushed into vials using a fritextractor. Ammonium hydroxide is added and the vials are sealed andheated at 65° C. overnight. All the oligonucleotides obtained were ofgood to high purity as shown by HPLC of their crude and of correctsequences as inferred by mass spectrometry. The quality and consistencyof all three-length nucleic acids were excellent.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. A single synthesis column produced by a process comprising: providinga cylinder having an open top and an open bottom end, embedding withinthe cylinder a cylindrical polyalkylene embedded silane-modified-CPGdevice, made by a process including: a) mixing an aqueous-freepolyalkylene with a silane-modified CPG; b) filling a cylindrical wellof an aluminum plate with said mixture; c) heating said plate at 180° C.to 220° C. for a predetermined schedule; and d) upon cooling, releasingfrom said plate said embedded device.
 2. The column according to claim1, in which the said polyalkylene is selected from the group consistingof ultrahigh molecular weight polyethylene, high density polyethylene,low density polyethylene, polypropylene, and mixtures thereof.
 3. Thecolumn according to claim 1, in which the quantity of the saidpolyalkylene is preferably from 50 to 70% by weight, based on the totalweight of the resin mixture.
 4. The column according to claim 1, inwhich silane-modified-CPG are controlled porous glass beads which havebeen modified with aminoalkyltrialkoxysilane,[alkylamino]alkyl(trialkoxy)silane or mercaptoalkyl(trialkoxy)silane andmixtures thereof.
 5. The column according to claim 1, wherein alkyl isselected from the group consisting of methyl, ethyl and propyl andwherein alkoxy is selected from the group consisting of methoxy, ethoxyand propoxy.
 6. The column according to claim 1, wherein the saidembedded devices contain less than ten micromoles of reactive amino ormercapto moieties.